Article Heterologous Systemic Prime–Intranasal Boosting Using a Spore SARS-CoV-2 Vaccine Confers Mucosal Immunity and Cross-Reactive Antibodies in Mice as well as Protection in Hamsters Paidamoyo M. Katsande 1 , Leira Fernández-Bastit 2,3 , William T. Ferreira 1,4 , Júlia Vergara-Alert 2,3 , Mateusz Hess 1 , Katie Lloyd-Jones 1 , Huynh A. Hong 1 , Joaquim Segales 2,5 and Simon M. Cutting 1, * 1 Department of Biological Sciences, Royal Holloway University of London, Egham TW20 0EX, UK 2 Unitat Mixta d’investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 3 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 4 SporeGen Ltd., London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK 5 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Spain * Correspondence: s.cutting@rhul.ac.uk; Tel.: +44-(0)7900-408-043 Abstract: Background: Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vac- Citation: Katsande, P.M.; cines are administered systemically and typically result in poor immunogenicity at the mucosa. As a Fernández-Bastit, L.; Ferreira, W.T.; result, vaccination is unable to reduce viral shedding and transmission, ultimately failing to prevent Vergara-Alert, J.; Hess, M.; infection. One possible solution is that of boosting a systemic vaccine via the nasal route resulting in Lloyd-Jones, K.; Hong, H.A.; mucosal immunity. Here, we have evaluated the potential of bacterial spores as an intranasal boost. Segales, J.; Cutting, S.M. Method: Spores engineered to express SARS-CoV-2 antigens were administered as an intranasal boost Heterologous Systemic following a prime with either recombinant Spike protein or the Oxford AZD1222 vaccine. Results: Prime–Intranasal Boosting Using a In mice, intranasal boosting following a prime of either Spike or vaccine produced antigen-specific Spore SARS-CoV-2 Vaccine Confers sIgA at the mucosa together with the increased production of Th1 and Th2 cytokines. In a hamster Mucosal Immunity and Cross-Reactive Antibodies in Mice as model of infection, the clinical and virological outcomes resulting from a SARS-CoV-2 challenge were Well as Protection in Hamsters. ameliorated. Wuhan-specific sIgA were shown to cross-react with Omicron antigens, suggesting that Vaccines 2022, 10, 1900. this strategy might offer protection against SARS-CoV-2 variants of concern. Conclusions: Despite https://doi.org/10.3390/ being a genetically modified organism, the spore vaccine platform is attractive since it offers biological vaccines10111900 containment, the rapid and cost-efficient production of vaccines together with heat stability. As such, Academic Editor: Rafael Blasco employed in a heterologous systemic prime–mucosal boost regimen, spore vaccines might have utility for current and future emerging diseases. Received: 11 October 2022 Accepted: 7 November 2022 Keywords: COVID-19; SARS-CoV-2; nasal vaccine; prime boost Published: 10 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. The coronavirus infectious disease 2019 (COVID-19) pandemic will be remembered both for its impact on global health and society but also for the remarkable speed at which prophylactic vaccines were implemented. Vaccines (mRNA, viral-vectored and subunit) have all shown high levels of protection against the most severe forms of disease, reducing Copyright: © 2022 by the authors. morbidity and mortality [1]. On the other hand, it has become apparent that current Licensee MDPI, Basel, Switzerland. COVID-19 vaccines are unable to prevent person-to-person transmission of its causal This article is an open access article distributed under the terms and agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with breakthrough conditions of the Creative Commons infections observed in fully vaccinated individuals supporting this [2,3]. The dissemination Attribution (CC BY) license (https:// of the virus will undoubtedly continue as a) immunity wanes and b) the reality that a creativecommons.org/licenses/by/ significant portion of the world’s population (~40–45%) has yet to receive their first vaccine 4.0/). dose [1]. The longer the virus continues to circulate, the greater the probability that new Vaccines 2022, 10, 1900. https://doi.org/10.3390/vaccines10111900 https://www.mdpi.com/journal/vaccines Vaccines 2022, 10, 1900 2 of 12 variants of concern (VOCs) will emerge [1]. The poor durability of current vaccines with protection of about six months may necessitate repeated booster campaigns. Taken together, the COVID-19 pandemic cannot yet be considered closed and will continue to represent a major burden to public health authorities until better vaccines are developed. SARS-CoV-2 transmits person-to-person by respiratory droplets and infects the upper respiratory tract (URT). Mucosal immunity is required to neutralize SARS-CoV-2 at the mucosal surfaces of the URT, either by tissue-resident memory T cells (TRMs) or a secretory IgA (sIgA) that can neutralize or prevent virus attachment within the epithelial cells of the mucosa. [4,5]. Vaccines administered by a parenteral route (noting that all COVID-19 vaccines so far are delivered by intra-muscular injection) fail to evoke a significant mucosal immune response, probably accounting for their failure to prevent the transmission and dissemination of the virus [6]. Accordingly, there is now a focus on developing mucosal COVID-19 vaccines of which intranasal is the optimal route of administration [7,8]. Mucosal vaccination has the potential to provide robust protective immunity at the mucosal site of pathogen entry by recruiting antigen-presenting cells (APCs) and/or engaging other innate immune cells. This requires safe and effective mucosal vaccination strategies that utilize both mucosal adjuvants and appropriate delivery systems [7]. SARS-CoV-2 vaccine peptides and a combination of flagellin (KF) and cyclic GMP-AMP (cgGAMP) as an adjuvant delivered intranasally have been shown to elicit strong systemic and mucosal immune responses that provide protection against SARS-CoV-2 challenge in a transgenic mouse model [9]. Early clinical data have suggested that without pre-existing immunity to SARS-CoV-2, immunity resulting from intranasal vaccination is suboptimal [7,10]. One promising approach might be that of boosting a current COVID-19 vaccine with an intranasal vaccine [11]. Adenoviral 5 and 19a vectored vaccines administered intranasally after a systemic mRNA vaccine prime were shown to induce high levels of both mucosal IgA and TRM , be fully protective in a mouse model of SARS-CoV-2 infection and neutralize VOCs [11]. Bacterial endospores of Bacillus subtilis engineered to display heterologous antigens have been shown to provide several advantages for mucosal immunization with efficacy against a number of bacterial and viral pathogens [12–14]. Spores carry natural mucosal adjuvant properties [15] and induce innate immunity [16]. Using a recently developed system (THY-X-CISE® ), engineered spores can be rendered unable to proliferate in the environment ensuring, biological containment [17]. Spores are of uniform size (1 µm diameter), can be produced rapidly in large quantities and are currently manufactured at medicinal grade by several pharmaceutical companies [18]. Most importantly, spores are heat stable (<70 ◦ C), and with a few exceptions, antigens expressed on their surface are stable both to heat and gastric enzymes [19]. Here, we engineered B. subtilis spores to express SARS-CoV-2 antigens and used these to nasally boost a systemic prime consisting of either a recombinant Spike protein or the Oxford AZD1222 vaccine. Our work shows that this heterologous systemic prime–mucosal spore boost strategy evokes mucosal immunity and is potentially protective. 2. Materials and Methods 2.1. General Methods Methods for B. subtilis, including the preparation of spores and the extraction of spore coat proteins, are described elsewhere [20]. B. subtilis strain PY79 is a prototrophic laboratory strain. 2.2. Construction of B. subtilis Spores Expressing SARS-CoV-2 Antigens A cloning method referred to as THY-X-CISE® [17] was used to introduce heterol- ogous genes into the chromosome of B. subtilis. First, chimeric genes were synthesized (Azenta Life Sciences), carrying the 50 segment (including promoter) of either the cotB or cotC genes of B. subtilis fused at their 30 -end to DNA-encoding SARS-CoV-2 domains. Gene sequences are shown in Supplementary Figure S1, which included the receptor- Vaccines 2022, 10, 1900 3 of 12 binding domain (RBD) (Wuhan-Hu-1 2019-nCov and Omicron B.1.1.529 variants) and the HR1-HR2 domain (Wuhan-Hu-1 2019-nCov) of the Spike protein. For clarity, these are referred to henceforth as SWuh , SOmi and HR1-HR2Wuh . Chimeras were then subcloned into the plasmid pThyA [17]. This placed the chimeric gene between the left and right arms of the thyA gene that encodes thymidine synthetase A (Supplementary Figure S1). The linearization of the resulting plasmid and introduction into the B. subtilis chromosome (strain PY79) by double-crossover recombination generated trimethoprim-resistant trans- formants that were thymine-dependent. The resulting thyA::insertion strain was then used as a recipient in a second transformation where an empty pThyB vector was introduced, thus disrupting the thyB locus (thyB::∆). The resulting strain (thyA::insertion thyB::∆) was thymine-dependent but resistant to a higher concentration of trimethoprim. The strains con- structed were PK120 thyA::cotB-RBDWuh thyB::∆, PK122 thyA::cotC-HR1-HR2Wuh thyB::∆ and PK230 thyA::cotB-RBDOmi thyB::∆. An isogenic strain, PK118 (thyA::∆ thyB::∆), which carries no gene insertions was made using the same procedure with empty pThyA and pThyB vectors that carry no gene insertions. PK118 (thyA::∆ thyB::∆), referred to henceforth as WT, was used as an isogenic control strain that produces “naked” spores devoid of SARS-CoV-2 antigens. Strains were confirmed by preparing spores, extracting spore coat proteins and probing Western blots with antibodies to the relevant SARS-CoV-2 domains: anti-SWuh PAbs raised against the entire SWuh polypeptide (SinoBiological, 40589-T62) and anti-SOmi MAbs that were raised against the RBD of the SOmi (SinoBiological, 40592-MM117). 2.3. Immunogens Recombinant Spike protein rSWuh and rSOmi (baculovirus expressed) were obtained from SinoBiological (Cat Nos. 40589-V08B1 and 40589-V08B33, respectively). Prior to intramuscular injection, 5 µg of protein was suspended in PBS and complexed (1:1) with the adjuvant AddaVax (Invivogen, vac-adx-10). The COVID-19 vaccine ChAdOx1 nCoV-19 (AZD1222) with a concentration of 3.8 × 109 infectious units (IU)/mL (3.7 × 1011 virus particles/mL) was provided by Prof. Sarah Gilbert (Jenner Institute, Oxford University) and referred to henceforth as AZD1222. Prior to use, AZD1222 was diluted in PBS to give a working concentration of 2 × 109 IU/mL. 2.4. Prime Boost Vaccination Studies BALB/c female mice were obtained from Charles River, UK (aged 8–10 weeks before study start) and allocated into groups (n = 5–6) for recombinant prime and n = 6 for the AZD1222 prime. (a) Recombinant prime boost vaccination (Supplementary Figure S2A) Group 1 received no immunizations and served as a naïve control group. Groups 2–4 each received an intramuscular injection (i.m.) of 50 µL (5 µg) of formulated rSWuh protein in each hind quadricep muscle. Group 2 was primed (i.m.) with rSWuh on day 1 and culled on day 48. Groups 3 and 4 were primed (i.m.) with rSWuh on day 1 and then intranasally (i.n.) boosted three and five weeks later with spores (1 × 109 CFU) of either WT (‘naked’) spores (Group 3) or a 1:1 mixture of PK120 (CotB-RBDWuh ) and PK122 (CotC-HR1-HR2Wuh ) referred to as SporCoVax (Group 4). Each intranasal administration (10 µL total/animal, 5 µL/nare, no anesthesia) consisted of three administrations given on consecutive days (Boost 1, days 21, 22 and 23 and Boost 2, days 35, 36 and 37). (b) AZD1222 prime boost vaccination (Supplementary Figure S2B) Group 1 was a naïve group and received no immunizations. Groups 2–5 each received 50 µL of AZD1222 (1.0 × 108 IU) in phosphate-buffered saline (PBS) in the right hind quadricep muscle. Group 2 was culled on day 48. Groups 3–5 were boosted (intranasal; 5 µL/nare, no anesthesia) three and five weeks later with spores (1 × 109 CFU) of WT (‘naked’; Group 3), SporCoVax (a 1:1 mixture of CotB-RBDWuh and CotC-HR1-HR2Wuh spores; Group 4) or PK230 (CotB-RBDOmi ; Group 5). Each intranasal administration (10 µL total/animal, 5 µL/nare, no anesthesia) consisted of three administrations given on consecutive days (Boost 1, days 21, 22 and 23 and Boost 2, days 35, 36 and 37). Vaccines 2022, 10, 1900 4 of 12 2.5. Determination of Mucosal and Systemic Antibody Titers by Indirect ELISA For the analysis of immunological responses, saliva was collected on days −1, 20, 34 and 48, serum was taken on day 48, and lungs were collected on day 48 and stored at −80 ◦ C. For lungs, sample extractions were made at a one-fifth (w/v) dilution in extraction buffer (2% (v/v) fetal calf serum) containing protease inhibitors, EDTA (0.05 mg/mL), as previously described [21]. Saliva extractions were made at a one-tenth (w/v) dilution in PBS. Samples were gently shaken for 2 h at 4 ◦ C to disrupt solid material, centrifuged (8000× g, 15 min.) and the supernatant used for analysis. Antibody levels in saliva (sIgA), lungs (IgA) and serum (IgG) were quantified by indirect enzyme-linked immunosorbent assay (ELISA). Greiner 96-well plates (MaxiSorp) were coated with 2 µg/mL of either rSWuh or rSOmi (50 µL/well) in PBS overnight at 4 ◦ C, followed by blocking for 1 h at RT with PBS containing 2% (w/v) bovine serum albumin (BSA). Saliva samples were diluted 1:20 in PBS. Lung and serum samples were diluted 1:10 and 1:1000, respectively, in diluent buffer (0.01 M PBS, 1% (w/v) BSA, 2% (v/v) FBS, 0.1% (v/v) Triton X-100, 0.05% (v/v) Tween 20). Samples were added to plates and 2-fold serially diluted. Plates containing saliva and lung samples were incubated for 2 h at 30 ◦ C, and those containing serum samples were incubated for 2 h at RT. Levels of IgA and IgG were detected using the appropriate horseradish peroxidase-conjugated anti-mouse IgA (Sigma Cat No. A4789-1) or anti-mouse IgG (Dako Cat No. P0447) in conjugate buffer (2% (v/v) FBS, 1% (v/v) BSA, 0.05% (v/v) Tween 20 in 0.01 PBS). Plates were incubated for 1 h at RT and then developed using tetramethyl benzidine (TMB) substrate (0.1 mg/mL 3.30 ,5.50 -tetramethylbenzidine in 0.1 M sodium acetate buffer (pH 5.5)). Reactions were stopped using 2 M H2 SO4 , and the ODs were read at 450 nm. Dilution curves were created for each sample, and endpoint titers were estimated as the maximum dilution that gave an absorbance reading above the average naïve sample. 2.6. Cytokine Analysis Mouse spleens were harvested, homogenized and treated with ACK lysis buffer (Sigma Cat. No 11814389001) to remove erythrocyte contamination. Splenocytes were cultured at 1 × 106 /well in 96-well plates. Cells were stimulated with 2.5 µg/mL of rSWuh for 72 h. IL-2, IL-5 and TNF-α in culture supernatants were measured using an 8-plex multiplex immunoassay (Biolegend Cat No. 741053) according to the manufacturer’s instructions. Data were acquired using a BD FACSCanto™ II, and analysis was performed using Legendplex software (Biolegend, San Diego, CA, USA). 2.7. Hamster Challenge Study Golden Syrian hamsters (age 6–8 weeks, 80–120 g) were allocated into groups (50% male, 50% females). Group 1 (n = 6) was a naïve, ‘unchallenged’ control group. Animals were dosed with PBS buffer on day 1 (i.m.), day 22 (i.n.) and day 35 (i.n.) and culled on day 49. Group 2 (n = 12) was a negative control group dosed as Group 1 but challenged with SARS-CoV-2 on day 42 and culled on day 49. Group 3 (n = 12) was primed (i.m.) on day 1 with rSWuh (10 µg, formulated 1:1 with the AddaVax adjuvant) and then boosted (intranasal) on days 22 and 35 with SporCoVax (a 1:1 mixture of CotB-RBDWuh and CotC-HR1-HR2Wuh spores at 2.5 × 109 CFU), after which, animals were challenged with SARS-CoV-2 on day 42 and culled on day 49. For intranasal administration, a total volume of 0.1 mL/dose (50 µL/nare) was used, and procedures were conducted under anesthesia (isofluorane). A SARS-CoV-2 Cat02 isolate (variant D614G, ID EPI_ISL-47147) was used for challenge. It was isolated from a patient in a laboratory-confirmed COVID-19 case in Barcelona, Spain, and propagated in Vero E6 cells (ATCC CRL-1586). The inoculum was prepared on the same day of the challenge at a concentration of 105 TCID50 /mL and kept refrigerated until use. Then, 0.1 mL was administered per challenge dose (50 µL/nare under isofluorane anesthe- sia), giving a challenge dose of 104 TCID50 . At 2 and 4 days post challenge, 4 animals/group were sacrificed for necropsy, and at day 7, all remaining animals were culled. Methods lenge. It was isolated from a patient in a laboratory-confirmed COVID-19 case in Barce- lona, Spain, and propagated in Vero E6 cells (ATCC CRL-1586). The inoculum was pre- pared on the same day of the challenge at a concentration of 105 TCID50/mL and kept re- frigerated until use. Then, 0.1 mL was administered per challenge dose (50 μL/nare under isofluorane anesthesia), giving a challenge dose of 104 TCID50. At 2 and 4 days post chal- Vaccines 2022, 10, 1900 5 of 12 lenge, 4 animals/group were sacrificed for necropsy, and at day 7, all remaining animals were culled. Methods for qPCR in nasal turbinates (NT), oropharyngeal (OP) swabs, etc., were performed as described elsewhere [22]. for qPCR in nasal turbinates (NT), oropharyngeal (OP) swabs, etc., were performed as described 2.8. elsewhere Statistical Analysis [22]. Statistical significance was assessed by Mann–Whitney U test. All statistical analyses 2.8. Statistical Analysis were performed using Prism (GraphPad, San Diego, CA, USA). Flow cytometry data were analyzedStatistical significance wasData using LEGENDplex™ assessed by Mann–Whitney Analysis Software. U test. All statistical analyses were performed using Prism (GraphPad, San Diego, CA, USA). Flow cytometry data were 3.analyzed Results using LEGENDplex™ Data Analysis Software. 3.1. Construction of B. subtilis Spores Displaying SARS‐CoV‐2 Domains 3. Results 3.1.Isogenic strains Construction ofsubtilis of B. B. subtilis were Spores engineered Displaying to expressDomains SARS-CoV-2 SARS-CoV-2 (Wuhan-Hu-1 variantIsogenic 2019-nCov)strainsantigens on thewere of B. subtilis spore surface astochimeric engineered fusions to the express SARS-CoV-2 spore coat (Wuhan-Hu-1 proteins variant 2019-nCov) antigens on the spore surface as chimeric fusions to the RBD CotB or CotC. This included two segments of the Spike (S) protein, the sporeand coat the HR1-HR2 domains fused to CotB (PK120, CotB-RBD Wuh) and CotC (PK122; CotC-HR1- proteins CotB or CotC. This included two segments of the Spike (S) protein, the RBD HR2 Wuh). A third strain, PK230, was made which expressed the RBD and the HR1-HR2 domains fused to CotB (PK120, CotB-RBDWuh of) the andOmicron vari- CotC (PK122; ant (B.1.1.529) CotC-HR1-HR2 fusedWuh to CotB (CotB-RBD Omi). ). A third strain, PK230, was made which expressed the RBD of the B. subtilis Omicron vaccine variant strains fused (B.1.1.529) carrying insertions to CotB at theOmi (CotB-RBD thyA). loci were examined by West- ern blotting of SDS-PAGE B. subtilis vaccine strains size-fractionated carrying insertions sporeatcoat proteins the thyA extracted loci were examinedfromby prepara- Western tions of pure blotting spores (approx. of SDS-PAGE 2 × 109 spores/extraction). size-fractionated Blots extracted spore coat proteins were probed fromwith PAbs or preparations MAbs spores (approx. 2 × 109 spores/extraction). Blots were probed with PAbs or as shown. of pure The use MAbs as shown. of anti-SWuh-specific PAbs Western blotting of proteins extracted from puri- fied spores of each The use of anti-S Wuh strain showed -specificthat PAbs theWestern RBD (primary blottingCotB-RBD of proteinsWuh species from extracted of ~62purified kDa) and HR1-HR2 spores of each(CotC-HR1-HR2 strain showed that Wuh , 40the kDa)RBDdomains (primarywere expressed CotB-RBD Wuhonspecies the sporeof surface. ~62 kDa) Proteins extracted and HR1-HR2 from spores of (CotC-HR1-HR2 Wuhan, isogenic WT strain 40 kDa) domains devoid were of SARS-CoV-2 expressed on the spore antigens surface. exhibited Proteins no cross-reaction extracted (Figure from spores of 1A). Using an an isogenic WT Omicron-specific strain devoid ofanti-S Omi MAb expres- SARS-CoV-2 antigens sion of RBDno exhibited was apparent in (Figure cross-reaction spores of PK230 1A). Usingas an a primary CotB-RBDanti-S Omicron-specific Omi MAb Omi species of ~63ex- pression of1B). RBD was apparent in spores of PK230 as a primary CotB-RBD Omi species of kDa (Figure ~63 kDa (Figure 1B). Figure 1. Spore coat expression of SARS-CoV-2 proteins. Panel (A) PK120 (thyA::cotB-RBDWuh ) Figure 1. Spore coat expression of SARS-CoV-2 proteins. Panel (A) PK120 (thyA::cotB‐RBDWuh) and and PK122 (thyA::cotC-HR1-HR2Wuh ) express the RBD and HR1-HR2 domains of Spike from the PK122 (thyA::cotC‐HR1‐HR2 ) express the RBD and HR1-HR2 domains of Spike from the Wuhan- Wuh Wuhan-Hu-1 (2019-nCoV) variant when probed with anti-S (Wuhan-Hu-1) PAbs. Bands corre- Hu-1 (2019-nCoV) variantWuhwhen probed with anti-S (Wuhan-Hu-1) PAbs. Bands corresponding to Wuh (40 kDa) are shown. No bands spondingWuhto(62 CotB-RBD CotB-RBD (62 kDa) andWuh kDa) and CotC-HR1-HR2 CotC-HR1-HR2 (40 kDa) are shown. No bands were detectable in were detectable spores in spores of the isogenic strainofWT the which isogenic strain WT expresses no which expresses heterologous no heterologous antigens. Panel (B) antigens. PK230 Panel (B) PK230 (thyA::cotB-RBDOmi ) expresses the RBD domain of Omicron (B.1.1.529) fused to CotB (63 kDa) when probed with anti-S (Omicron) PAbs. No bands were detectable in spores of the strain WT. 3.2. Intranasal Boosting with a Spore Vaccine following a Systemic Prime Evokes Mucosal Immune Responses We used a heterologous systemic prime–mucosal boost regimen to determine whether the intranasal administration of spore vaccines expressing SARS-CoV-2 antigens could evoke mucosal immunity. Mice were first dosed by intra-muscular injection (i.m.) with ei- ther the rSWuh protein or the AZD1222 vaccine and then, 21 and 35 days later, administered spore vaccines as an intranasal boost. For murine studies, a single dose consisted of three 3.2. Intranasal Boosting with a Spore Vaccine following a Systemic Prime Evokes Mucosal Im‐ mune Responses We used a heterologous systemic prime–mucosal boost regimen to determine whether the intranasal administration of spore vaccines expressing SARS-CoV-2 antigens could evoke mucosal immunity. Mice were first dosed by intra-muscular injection (i.m.) Vaccines 2022, 10, 1900 with either the rSWuh protein or the AZD1222 vaccine and then, 21 and 35 days later, 6ad- of 12 ministered spore vaccines as an intranasal boost. For murine studies, a single dose con- sisted of three daily intranasal administrations (e.g., days 21, 22 and 23) and resulted from daily the intranasaldosing regulatory administrations limitations(e.g., days 21, required for22mice. and 23) and resulted Following from the a prime of regulatory rSWuh or dosing limitations required for SporCoVax, mice. Following Wuh AZD1222 animals administered a 1:1 amixture prime ofofrSCotB-RBD or AZD1222 Wuh and animals CotC- administered SporCoVax, a 1:1 mixture of CotB-RBD Wuh Wuh , spores HR1-HR2 Wuh, spores exhibited antigen-specific sIgA in theand CotC-HR1-HR2 saliva together with serocon- exhibited version antigen-specific (Figure 2A,B). sIgA sIgA levelsinwere the saliva together(pwith significantly seroconversion < 0.001) greater than(Figure 2A,B). the group sIgA levels were significantly (p < 0.001) greater than the group receiving only an rS Wuh or receiving only an rS Wuh or AZD1222 prime, each of which yielded essentially no detectable AZD1222 sIgA (Figure prime, 2A,B).each of which yielded Interestingly, isogenic essentially no detectable spores (WT) carrying sIgA (Figure 2A,B). no SARS-CoV-2 Interest- antigens ingly, also isogenic evoked spores (WT)sIgA antigen-specific carrying nolevels but at SARS-CoV-2 antigens significantly loweralso (p